The catalytic polypeptides of diphtheria toxin, anthrax toxins (lethal factor [LF] and edema factor [EF]) and all serotypes of botulinum toxin are all delivered into the cellular cytosol by a process of endocytosis and passage through an acidic early endosomal compartment. Under acidic conditions the transmembrane domain of each of these toxins partially denature and spontaneously insert into the endosomal membrane forming a pore through which their respective catalytic domains are translocated. The molecular mechanism involved in catalytic domain translocation is the least well-understood step of this process. We have tentatively identified an "entry motif" on each of these toxins through BLAST analysis, and hypothesize that this "entry motif' acts through protein-protein interaction(s) with one or more proteins in a Cytosolic Translocation Factor (CTF) complex in the entry process. We have recently shown that a CTF complex is essential for the efficient transloaction of diphtheria toxin catalytic domain in vitro. Using analogous methods, we now propose to develop in vitro translocation assays for several of these toxins. We will use these assays and site-directed mutagenesis to determine whether the "entry motif' is indeed essential for entry, and, if so, which elements of the motif are most important. We will also use biochemical techniques to further define the necessary components of the CTF complex. If the entry motif hypothesis is valid, we will then be in a position to design a screen for small molecules that inhibit toxin action by specifically blocking catalytic domain translocation.